PROJECT SUMMARY Molecular mechanisms of the first-identified bacterial HORMA and Pch2-like proteins in a novel second- messenger signaling pathway In all cells, a variety of signaling pathways are responsible for sensing and responding to changes in internal environment, alterations in external conditions, and the presence of biological threats including infections. A major class of signaling pathway involves so-called 2nd-messenger molecules, many of which are cyclized forms of nucleotides (e.g. cyclic AMP) or di-nucleotides (e.g. cyclic di-AMP, cyclic di-GMP, and cyclic GMP-AMP). For bacteria in particular, nucleotide 2nd-messengers regulate a vast array of behaviors, allowing these organisms to quickly respond to changes in their environment, defend against phage threats, and initiate pathogenic behaviors when appropriate. Because of their importance to infectious and pathogenic behaviors within bacteria, plus growing evidence that nucleotide 2nd-messengers are sensed by hosts upon infection, bacterial 2nd-messenger signaling pathways are of high interest. Here, we propose to study the molecular mechanisms and biological roles of a novel signaling pathway recently discovered in a diverse set of environmental and pathogenic bacterial strains, which includes a 2nd-messenger synthase related to mammalian cGAS and bacterial DncV. This CD- NTase (cGAS/DcnV-like nucleotidyltransferases) is found in operons encoding the first-identified bacterial proteins containing the HORMA domain, a peptide-binding domain found in many critical signaling pathways in eukaryotes. The operons also encode an ortholog of the AAA+ ATPase Pch2, an important regulator of HORMA domain proteins. Our extensive preliminary data shows that the bacterial HORMA proteins form complexes with their cognate CD-NTases and are capable of dynamic conformational changes similar to their eukaryotic relatives. Moreover, these proteins bind specific peptides termed ?closure motifs?, suggesting that the CD- NTase/HORMA/Pch2 module constitutes a novel foreign-protein sensor. In this project, we will define optimal closure motif peptides that bind bacterial HORMA proteins, as a first step to identifying their biologically-relevant binding partners. We will next reconstitute 2nd-messenger synthesis by bacterial CD-NTases, and use mass spectrometry to identify the synthesized 2nd-messengers. We will use this assay to define how 2nd-messenger synthesis is regulated by HORMA proteins, their bound closure motifs, and Pch2. Finally, we will define the activities of a fourth protein encoded by this operon, a diverged nuclease-like protein that our data shows is allosterically activated by binding nucleotide 2nd-messengers. For all experiments, we will combine in vitro biochemistry and structure with cellular assays in a patient-derived strain of E. coli, where we propose that the operon functions as a novel restriction-modification or abortive infection pathway. Thus, we will not only define the molecular mechanisms of signaling by bacterial CD-NTases and their associated regulators, but will also outline one specific biological function for this pathway. This project will thereby set the stage for future work outlining the likely diverse roles of bacterial CD-NTases in bacterial conflict, defense, and pathogenesis.